In vivo high throughput toxicology screening method

ABSTRACT

A high throughput toxicology screening method is provided. In the subject method, at least 10 different compound compositions are tested simultaneously. Each compound composition is tested by contacting it with a plurality, e.g. from about 10 to 1000, of non-mammalian multi-cellular organisms and determining the effect of the compound composition on the organisms. The multi-cellular organisms employed in the subject methods are small, have differentiated tissues and organs and have a rapid generation time. The subject high throughput screening methods find use in a variety of applications, and are particularly suited for use in the toxicology screening of libraries of compounds, such as libraries of combinatorially produced compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C § 119 (e), this application is continuation of U.S.patent application Ser. No. 09/472,654, filed Dec. 27, 1999 and nowissued a U.S. Pat. No. 6,365,129; which application claims priority tothe filing date of the U.S. patent application Ser. No. 60/147,220 filedAug. 4, 1999, the disclosure of which is herein incorporated byreference.

INTRODUCTION

1. Field of the Invention

The field of this invention is toxicology testing, particularlytoxicology testing in pharmaceutical research and development.

2. Background of the Invention

During the drug development process, potential therapeutic agents ordrug candidates must be demonstrated to be both safe and effective fortheir intended use prior to obtaining FDA approval and subsequentcommercialization, at least in the United States. In drug developmentprocesses, potential drug candidates are subjected to mutagenicity andtoxicology assessments in an effort to demonstrate safety. Mutagenicityanalyses take place in bacteria (Ames test), Drosophila (Mueller-5test), and in mammalian cell culture. However, toxicology analyses arelimited to mammalian cell culture and animal model studies. This schemerequires significant time and money to be invested to analyze thetoxicity of a candidate drug. As such, toxicology studies are typicallyperformed after successful efficacy assessment for drug candidates.

With the advent of high throughput drug discovery, there is greatinterest in the pharmaceutical and related industries to streamline thetoxicology testing segment of the drug development process. As thenumber of drug candidates has exploded from 10's per year to 1,000's peryear, the toxicology assessment programs have become a severe bottleneckin the drug development process.

Accordingly, there is great interest in the development of new highthroughput screening assays which are capable of rapidly providingtoxicity data for a large number of different compounds. Of particularinterest would be the development of an in vivo high throughput toxicityscreening assay.

Relevant Literature

High throughput toxicity screening assays are discussed in: Kelly,“Advances in HTS Toxicology,” Genetic Engineering News, Mar. 1, 1999,pg. 14; and Sansome, Drug Discovery Today (1999) 4: 199-201.

SUMMARY OF THE INVENTION

High throughput toxicology screening assays are provided. In the subjectmethods a plurality of different compound compositions, usually at least10 different compound compositions, are simultaneously assayed for theirtoxic activity, if any. Each compound composition in the plurality isassayed for toxicity by contacting it with a population ofmulti-cellular organisms and determining the effect of the compoundcomposition on the multi-cellular organisms. Multi-cellular organismsthat find use in the subject high throughput screening (HTS) assays arethose that are small, have differentiated tissues and organs, have arapid generation time, and are prolific. The subject HTS methods finduse in a variety of applications, and are particularly suited for use inthe toxicological screening of large numbers of compounds, such ascombinatorially produced libraries of compounds.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

A high throughput toxicology screening method is provided. In thesubject method, at least 10 different compound compositions are testedsimultaneously. Each compound composition is tested by contacting itwith a plurality, e.g. from about 10 to 1000, non-mammalianmulti-cellular organisms and determining the effect of the compoundcomposition on the organisms. The multi-cellular organisms employed inthe subject methods are small, have differentiated tissues and organs,have a rapid generation time, and are prolific. The subject highthroughput screening methods find use in a variety of applications, andare particularly suited for use in the toxicology screening of librariesof compounds, such as libraries of combinatorially produced compounds.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms •a, ••an, “and •the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

As summarized above, the subject invention provides a high throughputscreening (HTS) method for simultaneously testing the toxicology of aplurality of different compound compositions. The subject HTS assays arein vivo toxicology assays, by which is meant that they determine theeffect of a compound on a living, multi-cellular organism. As such, thesubject HTS assays are distinguished from in vitro assays, in which cellcultures are employed.

In the subject HTS assays, a plurality of different compounds aresimultaneously tested. More specifically, a plurality of differentcompound compositions are simultaneously tested. Different compoundcompositions differ from each other in at least one of the followingcharacteristics: (a) they are made up of compounds that differ bymolecular formula; or (b) they are made up of compounds of the samemolecular formula but the compounds are present in differentconcentrations. In other words, any two given compound compositions aredifferent if they are either made up of compounds that differ bymolecular formula or are made up of the same type of compound but differwith respect to concentration of that compound. For example, a pluralityof different compound compositions may include 4 different types ofcompounds that differ by molecular formula, where each specific type ofcompound is present in three different concentrations, such that theplurality is made up of 12 different compound compositions.

By simultaneously tested is meant that each of the compound compositionsin the plurality are tested at substantially the same time. Thus, all ofthe compound compositions in the plurality are assayed for theirtoxicological effects in parallel. The number of compound compositionsin the plurality of compound compositions that are simultaneously testedis typically at least about 10, where in certain embodiments the numbermay be at least about 100 or at least about 1000, where the number ofcompound compositions tested may be higher. In general, the number ofcompound compositions that are tested simultaneously in the subject HTSmethods ranges from about 10 to 10,000, usually from about 100 to 10,000and in many embodiments from about 1000 to 5000.

In the subject methods, each individual compound composition in theplurality is individually assayed for toxicology. Each compoundcomposition is individually assayed for its toxicity by contacting thecompound composition with a plurality of non-mammalian multi-cellularorganisms and determining the effect of the compound composition (orlack thereof) on the organisms of the plurality. As the organismsemployed in the subject methods are multi-cellular, they includedifferentiated tissues and organs. They are further characterized bybeing relatively small, where by small is meant at least about 0.001 g,usually at least about 0.01 g and more usually at least about 0.1 g,where the average mass of each organism in the plurality may be as greatas 10 g or greater, but typically does not exceed about 100 g andusually does not exceed about 1,000 g. The multi-cellular organismsemployed in the subject HTS methods are also characterized by having arapid generation time. A rapid generation time is important to maintainthe breeding colony plus supply enough organisms that will be prolificenough to produce on average at least about 100 progeny per day, whichis the minimum requirement for high throughput sceening. For Drosophila,this minimum population of flies can range from 10 to 300, usually from50 to 150.

A number of different types of non-mammalian multi-cellular organismsmay be employed in the subject methods, where these types of organismsinclude insects, amphibians, fish, and the like. Specific organisms ofinterest include: Xenopus, Zebrafish, Caenerhabditis, Drosophila and thelike. Of particular interest in many embodiments are invertebrateanimals, particularly members of the phylum arthropoda, and moreparticularly members of the class insecta. Of particular interest inmany embodiments are flies. In many preferred embodiments, the flies aremembers of the family Drosophilidae, where the animal is often aDrosophila melanogaster. The multi-cellular organisms employed in thesubject invention may be at any stage of their life, e.g. in the larvalstage, in the adult stage, etc.

One specific multi-cellular organism of interest is a non-mammaliantransgenic animal having an adult onset neurodegenerative phenotype,e.g. a transgenic Drosophila melanogaster having an adult onsetneurodegenerative phenotype, as described in U.S. Patent ApplicationSer. No. 60/125,586, the disclosure of which is herein incorporated byreference. Another multi-cellular organism of particular interest is thenon-mammalian transgenic animal for cellular proliferative diseases,e.g. a transgenic Drosophila melanogaster having a neoplastic phenotype,as described in U.S. Patent Application Ser. No. 60/147,433 filed Aug.4, 1999, the disclosure of which is herein incorporated by reference.Also of interest are non-transgenic non-mammalian animals.

In the subject assay methods, each compound composition is brought intocontact with the population of multi-cellular organisms in a manner suchthat the active agent of the compound composition is capable of exertingactivity on at least a substantial portion of, if not all of, theindividual organisms of the population. By substantial portion is meantat least 40 number %, usually at least 50 number % and more usually atleast 60 number %, where the number % may be substantially higher and inmany embodiments can be as high as 80, 90 or 95 number % or higher.Generally, each compound agent is contacted with the members of thepopulation in a manner such that the active agent of the composition isinternalized by the organisms. Typically internalization will be byingestion, i.e. orally, such that that each compound composition willgenerally be contacted with the plurality of organisms by incorporatingthe compound composition in the nutrient medium, e.g. water, aqueoussolution of additional nutrient agents, etc., of the organisms. Forexample, where the multi-cellular organism is a fly, the candidate agentis generally orally administered to the fly by mixing the agent into thefly nutrient medium and placing the medium in the presence of the fly,(either the larva or adult fly, usually the larva) such that the flyfeeds on the medium.

As such, the compound composition may be contacted with the populationof multi-cellular organisms at any convenient stage during the lifecycle of the organism. Thus, depending on the particular organismsemployed, the compound composition is contacted with the organismsduring an immature life cycle stage, e.g. larval stage, during an adultstage, and the like.

A large number of different types of compounds may be assayed accordingto the subject invention. Compounds that may be assayed according to thesubject HTS methods encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Compounds generally comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecompounds often comprise cyclical carbon or heterocyclic structuresand/or aromatic or polyaromatic structures substituted with one or moreof the above functional groups. Compounds of interest are also foundamong biomolecules including, but not limited to: peptides, saccharides,fatty acids, steroids, purines, pyrimidines, derivatives, structuralanalogs or combinations thereof.

Compounds of interest are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. New potential therapeutic agents may also be createdusing methods such as rational drug design or computer modeling.

Screening may be directed to known pharmacologically active compoundsand chemical analogs thereof, or to new agents with unknown propertiessuch as those created through rational drug design.

Following contact of the compound composition with the population, theeffect of the compound on the members of the population is determined.The effect of the compound on the members of the population is generallydetermined by evaluating one or more of a number of different phenotypicparameters. Phenotypic parameters that are evaluated in a given HTSassay of the subject invention may vary widely depending, at least inpart, on the nature of the multi-cellular organisms being employed.Typically, phenotypic parameters that are evaluated in any given assayinclude one or more of the following: (1) viability; (2) morphologicaldefects; and (3) fecundity. Specific parameters that may be evaluatedinclude one or more of: (1) lethal dose, e.g. LD₅₀, LD₁₀ etc.); (2)growth defects; (3) sterility effect dose; (4) developmental defects;(5) neurologic impairment; (5) life-span modulation, e.g. life spanenhancing or shortening; and the like.

In addition to the above parameters that can be evaluated in the subjectmethods, the gene expression levels of the test organisms can beassayed, e.g. gene expression levels in treated larva, pupa, and/orflies can be evaulated. The genes can be from “houskeeping” genes thatprovide basic metabolic information to developmental and tissue specificgenes to gauge which tissue or cell type is affected and when. A varietyof different gene expression protocols, including arrays basedprotocols, are known to those of skill in the art, including thosedescribed in: EP 0 328 829 B1 and U.S. Pat. Nos. 5,468,613; 5,580,726;5,599,672; 5,512,462; 5,162,209 and 5,162,209, the disclosures of whichare herein incorporated by reference. Methods of analyzing differentialgene expression are also described in Maniatis, et al., MolecularCloning, A Laboratory Manual, (Cold Spring Harbor Press, Cold SpringHarbor, N.Y.)(1989); Nucleic Acid Hybridization, A Practical Approach(Hames, B. D., and Higgins, S. J. eds, IRL Press, Oxford)(1985); WO95/21944; Chalifour, et al., Anal. Biochem. (1994) 216: 299-304; Nguyenet al., Genomics (1995) 29: 207-216; Pietu et al., Genome Res. (1996) 6:492-503; and Zhao et al., Gene (1995) 166: 207-213.

The effect of the compound on the particular physical parameter orparameters being evaluated may be determined manually or robotically,such that in many embodiments determination of the effect of thecompound on the organism is accomplished via an automated procedure.

The effect of the compound on the phenotypic parameter or parameters isthen related to the toxicity of the compound. As such, the effect on thephenotypic parameter(s) is employed to derive a toxicity profile for theassayed compound, where toxicity profile refers to the toxic activity ofa given compound, i.e. its collection of one or more toxic activities,such as lethality, sterility causing activity, etc.

Because the subject methods are HTS methods in which a plurality ofcompounds are assayed for toxicity at the same time, any given HTSscreen according to the subject invention rapidly provides toxicityprofiles for a plurality of compounds. The number of compounds for whichtoxicity profiles are rapidly provided in any given HTS assay accordingto the subject invention ranges from about 20 to 50,000, usually fromabout 50 to 10,000 and more usually from about 500 to 5,000. As thetoxicity profiles are rapidly determined, they are determined ingenerally less than about 14 days, usually less than about 10 days. Inmany embodiments, they may be determined in less than about 7 days or ina shorter period of time.

In sum, the subject invention as described above provides a highthroughput method for toxicity screening of a large number of compoundsand/or different concentrations thereof. The subject HTS toxicityscreens find use in a variety of different applications in which it isdesired to obtain toxicity data for a large number of compounds in ashort period of time. Of particular interest in many embodiments is theuse of the subject methods to provide in vivo toxicity profiles forindividual compound members of libraries or collections of compounds,including combinatorially produced libraries of compounds. As such, thesubject HTS toxicology screening assays find use in a number ofapplications, including drug discovery and development applications.

The subject HTS methods may be part of a multi-step screening process ofevaluating candidate therapeutic agents for their efficacy (and safety)in the treatment of disease conditions. In multi-step screeningprocesses of the subject invention, a library of compounds is subjectedto screening in a second in vivo model, e.g. a mouse model, followingscreening via the subject HTS assays. Following the initial screening ofthe library using the HTS methods of the subject invention, the positivecompounds identified by the screen (i.e. those compounds that do nothave unacceptable toxicity profiles) are then screened in non-humanmammalian animal models, including transgenic non-human mammalian animalmodels, where the mammalian animal models are generally correlated tothe particular target disease condition. In addition, a pre in vivoscreening step may be employed, in which the library of compounds isfirst subjected to an in vitro screening assay for its potential as atherapeutic agent in the treatment of a particular disease condition.Any convenient in vitro screening assay may be employed, where a varietyof suitable in vitro screening assays are known to those of skill in theart, e.g. HTS cell culture assays.

The subject HTS toxicity screening assays also find use in thegeneration of databases of information that include toxicity profiles ofa plurality of distinct compounds. As such, the subject invention can beemployed to produce a database of toxicity profiles for a variety ofcompounds. Such a toxicity profile database will typically comprisetoxicity profile information as described above, for a number of relatedcompounds. The compounds of interest in a database may be selected andarranged according to various criteria: the types of molecules that aretested, e.g. steroids, antibiotics, antineoplastic agents, etc.; by thesource of compounds, e.g. environmental toxins, biologically activeextracts from a particular animal or cell, synthetic or natural libraryof compounds etc.; and the like.

The toxicity profiles and databases thereof may be provided in a varietyof media to facilitate their use. “Media” refers to a manufacture thatcontains the toxicity profile information of the present invention. Thedatabases of the present invention can be recorded on computer readablemedia, e.g. any medium that can be read and accessed directly by acomputer. Such media include, but are not limited to: magnetic storagemedia, such as floppy discs, hard disc storage medium, and magnetictape; optical storage media such as CD-ROM; electrical storage mediasuch as RAM and ROM; and hybrids of these categories such asmagnetic/optical storage media. One of skill in the art can readilyappreciate how any of the presently known computer readable mediums canbe used to create a manufacture comprising a recording of the presentdatabase information. “Recorded” refers to a process for storinginformation on computer readable medium, using any such methods as knownin the art. Any convenient data storage structure may be chosen, basedon the means used to access the stored information. A variety of dataprocessor programs and formats can be used for storage, e.g. wordprocessing text file, database format, etc.

As used herein, “a computer-based system” refers to the hardware means,software means, and data storage means used to analyze the informationof the present invention. The minimum hardware of the computer-basedsystems of the present invention comprises a central processing unit(CPU), input means, output means, and data storage means. A skilledartisan can readily appreciate that any one of the currently availablecomputer-based system are suitable for use in the present invention. Thedata storage means may comprise any manufacture comprising a recordingof the present information as described above, or a memory access meansthat can access such a manufacture.

A variety of structural formats for the input and output means can beused to input and output the information in the computer-based systemsof the present invention. One format for an output means ranks toxicityprofiles possessing varying degrees of similarity to a referencetoxicity profile. Such presentation provides a skilled artisan with aranking of similarities and identifies the degree of similaritycontained in the test toxicity profile.

The subject toxicity profile databases find use in a number of differentapplications. For example, where one has a compound of interest, one cansearch the database to determine whether that compound is present in thedatabase and, if so, readily identify the toxicity profile of thecompound. Alternatively, where one has a novel compound whose profile isnot present in the database, but one knows the structure of thecompound, one can search the database for similar compounds of similarstructure and obtain information regarding the toxicity of the compoundof interest through extrapolation. One can also compare a novel toxicityprofile of the a compound not present in the database with profilespresent in the database in order to identify compounds of similartoxicity to the compound of interest.

The comparison of a toxicity profile obtained from a test compound andtoxicity profiles present in the database, i.e. reference toxicityprofiles, is accomplished by any suitable deduction protocol, AI system,statistical comparison, etc. Methods of searching databases are known inthe art. See, for example, U.S. Pat. No. 5,060,143, which discloses ahighly efficient string search algorithm and circuit, utilizingcandidate data parallel, target data serial comparisons with an earlymismatch detection mechanism. For other examples, see U.S. Pat. Nos.5,720,009 and 5,752,019, the disclosure of which are herein incorporatedby reference.

Also provided by the subject invention are methods of screeninglibraries of compounds for their antitoxic effect, i.e. hight throughputmethods for the identification of antitoxin compounds. In such methods,a library of candidate antitoxin compounds is screened for activityaccording to the methods described above, where the test population ofanimals, e.g. flies, will have been previously contacted with the toxinfor which an antitoxin is sought or will be concomittantly contactedwith the toxin. Toxins of particular interest in many embodiments arethose that are toxic in both humans and the test animal, e.g. flies. Theanimal population is contacted with the toxin of interest using anyconvenient method, e.g. inclusion of the toxin in the nutrient medium,where the dose of toxin contacted with the population is a lethal dose,e.g. LD₉₅. Following contact of the test library of candidate antitoxinswith the animal population, those animals that survive are indicative ofthe animals that were given test compounds that have internalized anagent with the desired anti-toxin activity. Thus, also provided by thesubject invention is a high throughput method of screening for compoundshaving antitoxin activity.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

I. Validation of the Fly Model for Toxicology

In order to determine the extent that the fly model is valid fortoxicology assessment studies, an assessment of chemicals with knowntoxicological profiles and targets is generated using the fly. Thechemicals chosen have no substantial toxicity levels (e.g., food dye,sugar, etc.) to ones that are very toxic. The very toxic ones can bebroken down into three classes, ones that are generally toxic, ones thatare toxic to a cell, tissue, or organ type that is known to be in commonwith humans and flies, and ones that are toxic to a cell, tissue, ororgan type that is specific to humans.

A. Toxicity Tests

The chemical is dissolved in water at or near its saturation point.Serial dilutions of this stock solution are used to rehydrate instantfly media (Fisher Scientific). Specifically, one toxicity assay willcomprise of instant fly media rehydrated with pure stock solution of achemical, while another will be rehydrated with a 10% solution of thechemical (in water). This format will be used to generate data over a 4to 5 log dose range for each chemical tested.

A known quantity of embryos, typically between 40-50, is used as theinput. Specifically, 40 to 50 embryos are counted and placed in thereceptacle that contains the media/chemical mixture to be tested. Theembryos may be counted manually or by automation (e.g., liquidsuspension of embryos flowing through a diode). The larva feed on themedia/chemical mixture. All aspects of development from larva stage toadult must proceed normally in the presence of the chemical. The onlyfood and water source available to the larva and flies contains thechemical. It is shown that the variability of and intake amount that canbe expected using this protocol by feeding the larva chemicals that areeasy to assay. Specifically, Iron, Copper, and zinc have been selected.Sensitive and accurate kits are commercially available to analyze thesechemicals down to a concentration of 1 part per million. This willassign quantitative analyses to determine variability between larva in atest receptacle and between larva in different receptacles.

B. Toxicology Assessment

The developing larva and pupa are examined for normal growth anddevelopment. Then the adult flies are analyzed for lethality, sterility,developmental defects, and life span alterations. Lethality isdetermined by dividing the number of adult flies that eclose by thetotal number of embryos that were placed in the receptacle. Sterility isexamined for both males and females by crossing them to normal flies. Aphysical examination of the adults reveals any visible defects, such aslimb defects, tissue formation defects, abnormal coordination, etc.Finally the flies are allowed to live the natural span of their life todetermine whether an effect occurred to either shorten or lengthen theaverage lifespan of the fly.

II. Assays

A. Toxicity Screens

Using standard protocols, including the ones described above foradministration to flies, a variety of agents were administered atdifferent doses to mice, rats and flies and the LD for each agent wascompared. The results are provided in Table 1.

TABLE 1 Lethal dose toxicity trends are similar between mammals andflies. Mouse Rat Fly (oral LD50 (oral LD50 (oral LD50 Chemical Testedmg/ml) mg/ml) mg/ml) High toxicity sodium azide 27 27 .05 cadmiumchloride 60 88 .1 clonidine 135 126 .07 Moderate toxicity metrifonate300 560 .04 4-dimethyl n/a 250 .15 aminopyridine phenylenediamine 366510 .2 lithium chloride 1165 526 .4 Low toxicity disodium phosphate n/a17,000 142 niacinamide 2500 3500 5 polyvinyl pyrrolidol 40000 100000 <.4sodium borate 2000 2660 6.5 L-dopa 2363 1780 .7 bromophenol blue n/a n/a10

The trend for lower to higher toxic agents is similar in flies andmammals. The low toxics were categorized by having an LD50>1,500 mg/kgin mice and rats and these corresponded with a dose of greater than 0.4mg (the 0.4 mg dose for polyvinyl pyrrolidol was the maximum dose thatwas administerable to the flies and no toxicity was observed at thislevel). While the moderate range was defined as having a mouse or ratdose over 150 mg/kg up to 1400 mg/kg. This corresponded with a LD50 doserange of 0.15 to 0.4 for the fly. Finally the high toxic substances inmice and rats had an oral LD50 with less than 150 mg/kg. Thiscorresponded to the fly range that was 0.1 mg dose or less to induce anoral administered LD50. The one outlyer was metrifonate. This chemicalis of moderate toxicity in mice and rats, while it is clearly in thehigh toxic range for flies. As an initial high throughput screen, thispilot study suggests a >90% toxic assessment correspondence with miceand rats. This clearly demonstrates the potential to rapidly andaccurately estimate toxicity of unknown compounds.

B. AntiToxicity Screens

The activity of EDTA as an antitoxin was assessed by administering fliestoxins in conjunction with EDTA and without EDTA. The results areprovided in Table 2.

TABLE 2 EDTA is an effective in vivo anti-toxin in the fruit fly Flyfood Yeast Paste % survival 1.1 mM Copper None 0 6.0 mM Zinc None 0 Nochemical 25 mM EDTA 0 1.1 mM Copper 25 mM EDTA 76 6.0 mM Zinc 25 mM EDTA70 No chemical None 84 0.4 mM Copper None 50 2 mM Zinc None 50

The chemical EDTA binds to heavy metals, including copper and zinc. Thisbinding can occur in vitro or in vivo. In vivo binding can lead toneutralizing the toxicity of heavy metals to animals. However, sinceEDTA binds to calcium, it alone has a reasonable amount of toxicity. Theabove results demonstrate that a known heavy metal antitoxin can beidentified using the subject in animal antitoxin system. Shown in Table2 is the toxic effects of zinc, copper, and EDTA on the fruit fly. Thedata in Table 2 also show that the flies can survive in the prescence ofboth heavy metal (e.g., copper or zinc) and EDTA. To ensure that anyantitoxin effects from the EDTA would occur in vivo, the heavy metal andEDTA were supplied in two, physically separated food sources. The flieshad to eat from the fly food and yeast paste in the proper amount toneutralize the toxic effects, allowing for survival.

It is evident from the above results and discussion that the subjectinvention provides a valuable new high throughput screening method. Asthe subject methods employ multi-cellular organisms, they providevaluable in vivo toxicity data which is often better correlated toactivity in humans. Furthermore, the toxic effects on compounds can bedetermined on a number of different types of cells at substantially thesame time. In addition, the subject HTS assays are rapid. Accordingly,the subject invention represents a significant contribution to the art.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A high throughput toxicology screening method in which at least 10different compound compositions are simultaneously assayed for toxicity,said method comprising: simultaneously assaying at least 10 differentcompound compositions for toxicity, wherein each of said at least 10different compound compositions is assayed for toxicity by: (a)contacting said compound composition with a plurality of non-mammalianmulti-cellular organisms; and (b) determining the effect of saidcompound composition on said non-mammalian multi-cellular organisms toestimate the effect of said compound composition on a mammalian organismselected from a rat or a mouse.
 2. The screening method according toclaim 1, wherein said plurality of non-mammalian multi-cellularorganisms ranges from about 10 to
 1000. 3. The screening methodaccording to claim 1, wherein said non-mammalian multi-cellularorganisms have a rapid generation time.
 4. The screening methodaccording to claim 1, wherein said non-mammalian multi-cellularorganisms are small.
 5. The screening method according to claim 1,wherein said non-mammalian multi-cellular organisms are characterized bythe presence of differentiated organs and tissues.
 6. A high throughputtoxicology screening method in which at least 10 different compoundcompositions are simultaneously assayed for toxicity, said methodcomprising: simultaneously assaying at least 10 different compoundcompositions for toxicity, wherein each of said at least 10 differentcompound compositions is assayed or toxicity by: (a) contacting saidcompound composition with a population of from about 10 to 1000 smallnon-mammalian multi-cellular organisms having a rapid generation timeand differentiated organs end tissues; and (b) determining the effect ofsaid compound composition on said non-mammalian multi-cellular organismsto estimate the effect of said compound composition on a mammalianorganism selected from a rat or a mouse.
 7. The method according toclaim 6, wherein said population is characterized by producing at least100 progeny per day.
 8. The method according to claim 6, wherein atleast 100 compound compositions are tested simultaneously.
 9. The methodaccording to claim 6, wherein at least 1000 compound compositions aretested simultaneously.
 10. The method according to claim 6, wherein saidmulti-cellular organism is an insect.
 11. The method according to claim10, wherein said insect is a fly.
 12. A high throughput antitoxinscreening method in which at least 10 different candidate antitoxincompound compositions are simultaneously assayed for antitoxin activity,said method comprising: simultaneously assaying at least 10 differentcandidate antitoxin compound compositions for antitoxin activity,wherein each of said at least 10 different candidate compoundcompositions is assayed for antitoxin activity by: (a) contacting saidcandidate compound composition with a population of from about 10 to1000 small non-mammalian multi-cellular organisms having a rapidgeneration time and differentiated organs and tissues which have beencontacted with a toxin; and (b) determining the effect of said compoundcomposition on said non-mammalian multi-cellular organisms to estimatethe effect of said compound composition on a mammalian organism selectedfrom a rat or a mouse.
 13. The method according to claim 12, whereinsaid population is characterized by producing at least 100 progeny perday.
 14. The method according to claim 12, wherein at least 100candidate compound compositions are tested simultaneously.
 15. Themethod according to claim 6, wherein at least 1000 candidate compoundcompositions are tested simultaneously.
 16. The method according toclaim 6, wherein said multi-cellular organism is an insect.